Evaluating State Dependence and Subtype Selectivity of Calcium Channel Modulators in Automated Electrophysiology Assays.
ABSTRACT Abstract Voltage-gated Ca(2+) channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks™ Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCav1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug-channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB platform. The assays can be used to screen focused compound libraries for state-dependent Cav channel antagonists, to prioritize compounds for potency or to counterscreen for Cav subtype selectivity.
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ABSTRACT: Ion channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function. In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications. © 2014 Society for Laboratory Automation and Screening.Journal of Biomolecular Screening 12/2014; DOI:10.1177/1087057114560698 · 2.01 Impact Factor